Enterobacteria use a variety of compounds as terminal electron acceptors for respiration. Oxygen is used in preference to all other acceptors, and nitrate is the preferred anaerobic acceptor. Hierarchical regulation ensures that appropriate respiratory enzyme systems are synthesized in response to changing availability of electron acceptors. Thus, during anaerobic growth, nitrate induces synthesis of formate dehydrogenase-N and nitrate reductase, and represses synthesis of respiratory enzymes that metabolize less preferred electron acceptors, such as nitrite and fumarate. Previous work supported by this grant has identified interacting two component regulatory systems that control transcriptional regulation of respiratory enzyme synthesis in response to nitrate and its reduction product, nitrite. Dual sensors (the NARX and NARQ proteins) monitor the availability of nitrate and nitrite, and phosphorylate or dephosphorylate dual response regulators (the NARL and NARP proteins) accordingly. The long-term objective of this research is to understand the action of these parallel regulatory systems to coordinate gene expression in response to nitrate and nitrite availability. The cis-acting regulatory sites for the NARL-activated narG and fdnG operons have been previously characterized. This research will characterize the cis-acting sites for a NARP-activated operon, aeg-46.5. Comparison with the NARL-activated regulatory sequences will reveal similarities and differences in DNA binding by the homologous response regulators, NARL and NARP. Mutational and biochemical analysis of the sensors NARX and NARQ, and of the response regulators NARL and NARP, will help define sensor-response regulator interactions involved in both positive and negative regulation. Additionally, this analysis will probe sensor recognition of signal molecules (nitrate and nitrite), and response regulator recognition of specific DNA binding sequences. The NARL and NARP proteins will be analyzed in vitro with respect to specific protein-DNA interactions. Finally, the physiology of nitrate and nitrite signaling will be dissected by monitoring the rate of target operon induction or repression in response to varying concentrations and ratios of the signal molecules, nitrate and nitrite. Overall, this research will advance our understanding of both anaerobic physiology and metabolism, and of bacterial signal transduction pathways.